Targefrin: A Potent Agent Targeting the Ligand Binding Domain of EphA2

Overexpression of the receptor tyrosine kinase EphA2 is invariably associated with poor prognosis and development of aggressive metastatic cancers. Guided by our recently solved X-ray structure of the complex between an agonistic peptide and EphA2-LBD, we report on a novel agent, targefrin, that binds to EphA2-LBD with a 21 nM dissociation constant by isothermal titration calorimetry and presents an IC50 value of 10.8 nM in a biochemical assay. In cell-based assays, a dimeric version of the agent is as effective as the natural dimeric ligands (ephrinA1-Fc) in inducing cellular receptor internalization and degradation in several pancreatic cancer cell lines. When conjugated with chemotherapy, the agents can effectively deliver paclitaxel to pancreatic cancers in a mouse xenograft study. Given the pivotal role of EphA2 in tumor progression, we are confident that the agents reported could be further developed into innovative EphA2-targeting therapeutics.

• Supplementary Figure S1 reports the synthetic scheme used to prepare monomeric peptides. Page S7 • Supplementary Figure S2 reports the synthetic scheme used to prepare dimeric peptides.
Page S8 • Supplementary Figure S3 reports the synthetic scheme used to prepare targefrin-motif.
Page S9 • Supplementary Figure S4 reports the synthetic scheme used to prepare targefrin-dimermotif. Page S10 • Supplementary Figure S5 reports the synthetic scheme used to prepare targefrin-PTX.Page S11 • Supplementary Figure S6 reports the synthetic scheme used to prepare targefrin-dimer-PTX. Page S12 • Supplementary Figure S7 reports the synthetic scheme used to prepare targefrin-dimer-TAMRA. Page S13 • Supplementary Figure S8 reports HPLC traces of keys compounds. Page S14 • Supplementary Figure S9 reports pharmacokinetics studies. Page S16 • Supplementary Figure S10 reports uncropped western blots that generated the data in Figures 4, 5 and 8. Page S17 • Supplementary Figure S11 reports cell migration assay of BxPC3 at 12 h (relative to Figure 9). Page S20 • Supplementary Figure S12 reports cell viability assay of MIA PaCa-2 treated with targefrin or ephrinA1-Fc. Page S21 • Supplementary Figure S13 reports chemistry analysis for mice treated with targefrindimer. Page S22 • Supplementary Figure S14 reports repeated doses toxicity studies with targefrin-dimer-PTX versus PTX alone. Page S23 Table S1. Mass-spectrometry data of investigated compounds. All the compounds were analyzed using an Agilent 6545 QTOF LC/MS instrument.

Targefrin-PTX
Targefrin-dimer-PTX Figure S9. Pharmacokinetics studies. Preliminary pharmacokinetic (PK) studies with Targefrin-dimer. The agent has been injected IV via the tail vein at a concentration of 50 mg/Kg in a formulation of 80% PBS, 10% Tween 80, and 10% Ethanol. Note that is formulation resulted in a clear solution containing 20mg/ml of targefrin-dimer. Cmax ~ 650 ng/mL after 2 hours from the injection. Estimated t1/2 ~ 15 hr. S17 Figure S10. Duplicate experiments reporting uncropped western blots that generated the data in Figures 4, 5 and 8. Figure S11. Cell migration assay of BxPC3 from Figure 9 at 12 h. A) Cell migration assay of BxPC3 treated with 2 µg/mL ephrinA1-Fc and 10 µM targefrin or the indicated doses of targefrin-dimer. The yellow lines displayed initial scratches made at 0 h while the black lines displayed the location that the cells had migrated to after 12 h. B) Targefrin-dimer significantly inhibited cell migration at 12 h in a dosedependent manner as shown by decreases in relative wound density. ***p < 0.01, ****p < 0.0001, as determined by a one-way analysis of variance using Dunnett's post-test analysis. Figure S12. Cell viability assay of MIA PaCa-2 at 72 h. A) MIA PaCa-2 cells were treated with 1 µg/mL ephrinA1-Fc, different doses of Targefrin or Targefrin-dimer for 72 h. Percent confluency was monitored with the IncuCyte S3 live-cell analysis system and percent cell viability was calculated by normalizing the confluency of the treatments to that of the DMSO control. Percent cell viability was not significantly affected across all the treatments, as determined by a two-way analysis of variance using Bonferroni post-test analysis. B) Time-response curves for the percent confluency of MIA PaCa-2 cells after the indicated treatments.  Figure S13. Blood chemistry analysis for mice treated with Targefrin-dimer. 5 Balb/c mice were treated with Targefrin-dimer 20 mg/mL (80% PBS, 10% Tween 80, 10% Ethanol) to obtain 50 mg/kg doses. After 24 hr mice were sacrificed and full chemistry panel analysis was conducted as listed below.

Figure S14: Repeated doses toxicity studies with Targefrin-dimer-PTX versus PTX alone.
Balc/c mice received equimolar doses of PTX or targefrin-dimer-PTX daily (IV), and body weight were measured daily. FD = found dead. By day 5 all 3 mice in the PTX treated group were found dead. Mice treated with targefrin-dimer-PTX were lethargic after the first doses but recovered. No signs of toxicity were noted in the mice treated with targefrin-dimer.